Chemotaxis by the RAW264 mouse macrophage cell line was inhibited by 3-deazaadenosine but not by 3-deazaaristeromycin. A search for biochemical reactions inhibited by 3-deazaadenosine but not by 3-deazaaristeromycin has revealed that only one reaction, the synthesis of a small number of proteins identified after separation by two-dimensional polyacrylamide gel electrophoresis, has the necessary inhibitor specificity for involvement in the 3-deazaadenosine-sensitive step of chemotaxis. A study with several adenosine analogs showed a correlation betwen inhibition of chemotaxis and inhibition of the synthesis of a common subset of proteins. These analogs also inhibited the synthesis of polyadenylated mRNA, leading us to postulate that incubation of cells with 3-deazaadenosine inhibits a methylation reaction that is required for the formation of a functional mRNA coding for one or more proteins required for chemotaxis. Experiments to identify attractant-specific proteins have been limited because chemically defined attractants for RAW264 cells have not been available. This problem has been overcome by the isolation of a stable cell hybrid from a fusion betwen human leukocytes and a thioguanineresistant RAW264 cell line. The hybrid expressed functional genes for chemotaxis to N-formylmet-leu-phe, a commercially available synthetic attractant. The hybrid has been used to show that chemotaxis is inhibited by pertussis toxin. For inhibition of chemotaxis, pertussis toxin must enter the cell and ADP-ribosylate a 41,000 molecular weight protein. Studies of the inhibition of chemotaxis by pertussis toxin and cholera toxin have led us to conclude that guanine nucleotide binding proteins are involved in transduction of the chemotactic signal. Guanine nucleotide binding proteins do not couple chemoattractant receptors to adenylate cyclase. This conclusion is based on the finding that there is no correlation betwen inhibition of chemotaxis and levels of cAMP and on the observation that attractants do not change cAMP levels or alter the activity of adenylate cyclase.